Electroneutral Na+-Cl- cotransporters [including bumetanide-sensitive Na+:K+:2Cl- and thiazide-sensitive Na+:Cl- cotransporters] comprise a newly recognized and distinct family of proteins that participate in epithelial salt absorptive and secretory processes, in cell volume regulation, in the early response of cells to mitogenic factors, and in the response of vascular endothelial and smooth muscle cells to vasoactive agents. In the mammalian kidney the bumetanide-sensitive, Na+:K+:2Cl- cotransporter [BSC] is most abundant on apical membranes of the thick ascending limb of Henle's loop [TAL] where it mediates NaC1 absorption, a process vital to urine dilution and concentration. Our recent cloning of the BSC [BSC-r1] from rate outer medulla has now provided the basis for studying the structure, function and regulation of this important transporter at a molecular level. The ion and diuretic inhibitor kinetics, and the factors regulating activity, of the BSC-r1 cotransporter will be determined in oocytes, isolated TAL tubules and stably transfected cells. The localization of the cotransporter gene products in the rat kidney will be assessed by Northern analysis, in situ hybridization and PCR of single tubules. Polyclonal antibodies produced against the cotransporter will be used to detect cotransporter protein in rat kidney using Western analysis and immunocytochemistry. The role of phosphorylation-dephosphorylation in cotransporter function will be studied. Related gene products will be identified in rat and mouse kidney by low stringency screening of cDNA libraries and by PCR. The functional characteristics of these related cDNAs will be determined in X. laevis oocytes. Site-directed mutagenesis and chimera constructs will be used in combination with isotopic flux studies and electrical current measurements to begin to identify the molecular regions influencing Na+, K+ and Cl- binding/translocation and phosphorylation of the BSC-r1 protein. The results of these studies will provide a molecular basis for understanding Na+:K+:2Cl- cotransporter function and regulation in health and disease.